ABSTRACT During development, a single human genome gives rise to hundreds of differentiated cell types that must maintain their distinct identities. Proteins and complexes of the Polycomb group modify chromatin chemically and physically and are required to maintain repression of lineage-specific genes in inappropriate cell types. The Polycomb repressive complex 2 (PRC2) interprets the transcriptional and epigenetic state of the nucleus and trimethylates histone H3 at lysine 27 (H3K27me3), which imposes chromatin-based silencing. The self- propagating nature of H3K27me3 explains the epigenetic maintenance of these silent chromatin domains once established, and therefore the maintenance of appropriate cell identities; however, different genes are silenced by PRC2 in different lineages, suggesting that at critical junctures in development, PRC2 must be able to select new genes to be repressed. The goal of this proposal is to decipher the molecular logic that controls the establishment of new PRC2- silenced chromatin domains during development. Specifically, we will test the hypothesis that the two PRC2 complex types, PRC2.1 and PRC2.2 silence different genes during development due to their distinct accessory protein subunits and interacting RNAs. We will test this hypothesis with two specific aims. In Aim 1, we will utilize a state-of-the art inducible protein degradation system to discern the roles of PRC2.1 and PRC2.2 at two critical steps of early development, the transition from ground to primed pluripotency and the commitment to the neural lineage. We will induce degradation of accessory subunits that define the two complex types and analyze molecular and functional phenotypes at different time points during the directed differentiation of embryonic stem cells into neuronal progenitors. The reversibility of the protein degradation system will allow us to restore the complexes during or after differentiation and determine the exact moment at which their function is required. In Aim 2, we will follow up on our recently published work that identified multiple RNA-binding protein surfaces on both PRC2.1 and PRC2.2 and use this information to design separation-of-function RNA-binding mutants. With these mutants, we will identify RNAs bound to the different subunits of PRC2.1 and PRC2.2. Next, we will utilize inducible protein degradation followed by rescue with RNA-binding mutants to determine how RNA interactions contribute to PRC2.1 and PRC2.2 recruitment and function on chromatin. The proposed studies will provide insight on the molecular mechanisms that underpin the creation of new silent chromatin regions by PRC2 during differentiation, with broad implications for our understanding of epigenetic processes during normal development and their dysregulation in disease.